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The process of introducing nucleic acids into eukaryotic cells by nonviral methods is
defined as transfection. Using various chemical, lipid or physical methods, this gene
transfer technology is a powerful tool to study gene function and protein expression in
the context of a cell. Development of reporter gene systems and selection methods for
stable maintenance and expression of transferred DNA have greatly expanded the
applications for transfection. Assay-based reporter technology, together with the
availability of transfection reagents, provides the foundation to study mammalian
promoter and enhancer sequences, trans-acting proteins such as
transcription factors, mRNA processing, protein:protein interactions, translation and
recombination events (Groskreutz and Schenborn, 1997).

Transfection is a method that neutralizes or obviates the issue of introducing
negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with
a negatively charged membrane. Chemicals like calcium phosphate and DEAE-dextran or
cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall
positive charge to the molecule
(Figure 12.1). This makes it easier for the DNA:transfection reagent complex to
cross the membrane, especially for lipids that have a “fusogenic” component, which
enhances fusion with the lipid bilayer. Physical methods like microinjection or
electroporation simply punch through the membrane and introduce DNA directly into the
cytoplasm. Each of these transfection technologies is discussed in the following
sections.

Figure 12.1. Schematic representation of various transfection technologies and how they
neutralize the negatively charged DNA.Note that lipid-based reagents also can coat DNA in addition to forming
micelles and associating with DNA by attraction as depicted.

This chapter covers general information on transfection techniques and
considerations for transfection efficiency and optimization. In addition, we discuss
various transfection agents available from Promega as well as general protocols for
transfection and specific examples using our transfection reagents. Finally, we review
stable transfection and outline a protocol using drug selection.

One of the first chemical reagents used to transfer nucleic acids into cultured
mammalian cells was DEAE-dextran (Vaheri and Pagano, 1965; McCutchan and Pagano,
1968). DEAE-dextran is a cationic polymer that tightly associates with negatively
charged nucleic acids. An excess of positive charge, contributed by the polymer in
the DNA:polymer complex, allows the complex to come into closer association with the
negatively charged cell membrane. Uptake of the complex is presumably by endocytosis.
This method successfully delivers nucleic acids into cells for transient expression;
that is, for short-term expression studies of a few days in duration. However, this
technique is not generally useful for stable or long-term transfection studies that
rely upon integration of transferred DNA into the chromosome (Gluzman, 1981). Other
synthetic cationic polymers have been used to transfer DNA into cells, including
polybrene (Kawai and Nishizawa, 1984), polyethyleneimine (Boussif et
al. 1995) and dendrimers (Haensler and Szoka, 1993; Kukowska-Latallo
et al. 1996).

Calcium phosphate co-precipitation became a popular transfection technique
following the systematic examination of this method in the early 1970s (Graham and
van der Eb, 1973). The authors examined the performance of various cations and
effects of cation concentration, phosphate concentration and pH on transfection.
Calcium phosphate co-precipitation is widely used because the components are easily
available and inexpensive, the protocol is easy-to-use, and it is effective with many
different types of cultured cells. The protocol involves mixing DNA with calcium
chloride, adding this in a controlled manner to a buffered saline/phosphate solution
and allowing the mixture to incubate at room temperature. The controlled addition
generates a precipitate that is dispersed onto the cultured cells. The precipitate is
taken up by cells via endocytosis or phagocytosis. Calcium phosphate transfection is
routinely used for both transient and stable transfection of a variety of cell types.
In addition, calcium phosphate appears to provide protection against intracellular
and serum nucleases (Loyter et al. 1982).

However, calcium phosphate co-precipitation is prone to variability and is not
suited for in vivo gene transfer to whole animals. In addition, small pH changes (±
0.1) can compromise the efficacy of calcium phosphate transfection (Felgner, 1990).
Promega offers the calcium phosphate reagent as part of the
ProFection® Mammalian Transfection System—Calcium
Phosphate (Cat.# E1200).

The term “liposome” refers to lipid bilayers that form colloidal particles in an
aqueous medium (Sessa and Weissmann, 1968). By 1980, artificial liposomes were being
used to deliver DNA into cells (Fraley et al. 1980). The next
advance in liposomal vehicles was development of synthetic cationic lipids by Felgner
and colleagues (Felgner et al. 1987). The cationic head group of
the lipid compound associates with negatively charged phosphates on the nucleic acid.
Liposome-mediated delivery offers advantages such as relatively high efficiency of
gene transfer, ability to transfect certain cell types that are resistant to calcium
phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of
DNA of all sizes from oligonucleotides to yeast artificial chromosomes (Felgner
et al. 1987; Capaccioli et al. 1993;
Felgner et al. 1993; Haensler and Szoka, 1993; Lee and Jaenisch,
1996; Lamb and Gearhart, 1995), delivery of RNA (Malone et al.
1989; Wilson et al. 1979), and delivery of protein (Debs
et al. 1990). Cells transfected by liposome techniques can be
used for transient expression studies and long-term experiments that rely on
integration of DNA into the chromosome or episomal maintenance. Unlike DEAE-dextran
or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be
used for in vivo transfer of DNA and RNA to animals and humans (Felgner et
al. 1995).

A lipid with overall net positive charge at physiological pH is the most common
synthetic lipid component of liposomes developed for gene delivery (Figure 12.2).
Often the cationic lipid is mixed with a neutral lipid such as
L-dioleoyl phosphatidylethanolamine (DOPE; Figure 12.3), which
can enhance the gene transfer ability of certain synthetic cationic lipids (Felgner
et al. 1994; Wheeler et al. 1996). The
cationic portion of the lipid molecule associates with negatively charged nucleic
acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex
(Kabanov and Kabanov, 1995; Labat-Moleur et al. 1996),
presumably from electrostatic interactions between the negatively charged nucleic
acid and positively charged head group of the synthetic lipid. For cultured cells, an
overall net positive charge of the liposome/nucleic acid complex generally results in
higher transfer efficiencies, presumably because this allows closer association of
the complex with the negatively charged cell membrane. Entry of the liposome complex
into the cell may occur by endocytosis or fusion with the plasma membrane via the
lipid moieties of the liposome (Gao and Huang, 1995). Following cellular
internalization, the complexes appear in the endosomes and later in the nucleus. It
is unclear how the nucleic acids are released from the endosomes and lysosomes and
traverse the nuclear membrane. DOPE is considered a “fusogenic” lipid (Farhood
et al. 1995), and its role may be to release these complexes
from endosomes as well as to facilitate fusion of the outer cell membrane with
liposome/nucleic acid complexes. While DNA will need to enter the nucleus, the
cytoplasm is the site of action for RNA, protein or antisense oligonucleotides
delivered via liposomes.

Figure 12.2. The general structure of a synthetic cationic lipid.X, Y and Z represent a number of possible chemical moieties, which can
differ, depending on the specific lipid.

Figure 12.3. Structure of the neutral lipid DOPE.

Promega offers the FuGENE® HD Transfection Reagent
(Cat.# E2311), a novel nonliposomal transfection
reagent with wide application in different cell types and low toxicity,
FuGENE® 6 Transfection Reagent (Cat.#
E2691, a nonliposomal formulation that transfects a wide variety
of cell lines with high efficiency and low toxicity, and the TransFast™ Transfection
Reagent (Cat.# E2431), which uses a polycationic head
group attached to a lipid backbone structure to deliver nucleic acids into eukaryotic
cells. The best transfection reagent and conditions for a particular cell type must
be empirically and systematically determined because inherent properties of the cell
influence the success of any specific transfection method.

Physical methods for gene transfer were developed and used beginning in the early
1980s. Direct microinjection into cultured cells or nuclei is an effective although
laborious technique to deliver nucleic acids into cells by means of a fine needle
(Cappechi, 1980). This method has been used to transfer DNA into embryonic stem cells
that are used to produce transgenic organisms (Bockamp et al.
2002) and to introduce antisense RNA into C. elegans (Wu
et al. 1998). However, the apparatus is costly and the
technique extremely labor-intensive, thus it is not an appropriate method for studies
that require a large number of transfected cells.

Electroporation was first reported for gene transfer studies in mouse cells (Wong
and Neumann, 1982). This technique is often used for cell types such as plant
protoplasts, which are difficult to transfect by other methods. The mechanism is
based on the use of an electrical pulse to perturb the cell membrane and form
transient pores that allow passage of nucleic acids into the cell (Shigekawa and
Dower, 1988). The technique requires fine-tuning and optimization of pulse duration
and strength for each type of cell used. In addition, electroporation often requires
more cells than chemical methods because of substantial cell death, and extensive
optimization often is required to balance transfection efficiency and cell viability.
More modern instrumentation allows nucleic acid delivery to the nucleus and
successful transfer of DNA and RNA to primary and stem cells.

Another physical method of gene delivery is biolistic particle delivery, also
known as particle bombardment. This method relies upon high-velocity delivery of
nucleic acids on microprojectiles to recipient cells by membrane penetration (Ye
et al. 1990). This method is successfully employed to deliver
nucleic acid to cultured cells as well as to cells in vivo (Klein et
al. 1987; Burkholder et al. 1993; Ogura et
al. 2005). Biolistic particle delivery is relatively costly for many
research applications, but the technology also can be used for genetic vaccination
and agricultural applications.

While transfection has been used successfully for gene transfer, the use of
viruses as vectors has been explored as an alternative method to deliver foreign
genes into cells and as a possible in vivo option. Adenoviral vectors are useful for
gene transfer due to a number of key features: 1) they rapidly infect a broad range
of human cells and can achieve high levels of gene transfer compared to other
available vectors; 2) adenoviral vectors can accommodate relatively large segments of
DNA (up to 7.5kb) and transduce these transgenes in nonproliferating cells; and 3)
adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques
(Vorburger and Hunt, 2002). Other vectors of interest include adeno-associated virus,
herpes simplex virus, retroviruses and lentiviruses, a subset of the retrovirus
family. Lentiviruses (e.g., HIV-1) are of particular interest because they are well
studied, can infect quiescent cells, and can integrate into the host cell genome to
allow stable, long-term transgene expression (Anson, 2004).

As with all gene transfer methods, there are drawbacks. For adenoviral vectors,
packaging capacity is low, and production is labor-intensive (Vorburger and Hunt,
2002). With retroviral vectors, there is the potential for activation of latent
disease and, if there are replication-competent viruses present, activation of
endogenous retroviruses and limited transgene expression (Vorburger and Hunt, 2002;
Anson, 2004).

With so many different methods of gene transfer, how do you choose the right
transfection reagent or technique for your needs? Any time a new parameter, like a
new cell line, is introduced, the optimal conditions for transfection will need to be
determined. This may involve choosing a new transfection reagent. For example, one
reagent may work well with HEK-293 cells, but a second reagent is a better choice
when using HepG2 cells. Promega offers the FuGENE® HD Protocol Database to help identify
a protocol for your cell line when using the FuGENE® HD
Transfection Reagent. A drop-down menu allows you to search the database by cell
line, plate type and number of cells to be transfected. The Transfection Assistant
also offers a drop-down menu to select cell lines and either
FuGENE® HD or FuGENE® 6
Transfection Reagent for transfection conditions. The conditions should be considered
only guidelines since you may need to optimize the transfection conditions for your
specific application. See Optimization of Transfection
Efficiency (Section IV) and General Transfection
Protocol (Section VI) for details.

Another parameter to consider is the time frame of the experiment you wish to
conduct. Is it short- or long-term? For instance, determining which promoter deletion
constructs can still function as a promoter can be accomplished with a transient
transfection experiment, while establishing stable expression of an exogeneously
introduced gene construct will require a longer term experiment.

Transient Expression

Cells are typically harvested 24–72 hours post-transfection for studies
designed to analyze transient expression of transfected genes. The optimal time
interval depends on the cell type, research goals and specific expression
characteristics of the transferred gene. Analysis of gene products may require
isolation of RNA or protein for enzymatic activity assays or immunoassays. The
method used for cell harvest will depend on the end product assayed. For example,
expression of the firefly luciferase gene in the
pGL4.10[luc2] Vector (Cat.#
E6651) is generally assayed 24–48 hours post-transfection, whereas
the pGL4.12[luc2CP] Vector (Cat.#
E6671) with its protein degradation sequences can be assayed in a
shorter time frame (e.g., 3–12 hours), depending on the research goals and the
time it takes for the reporter gene to reach steady state. For more information on
luminescent reporter genes like firefly luciferase, see the Protocols
and Applications Guide chapter on bioluminescent reporters.

When performing a transient transfection, you can choose between a standard or
reverse transfection protocol. In a standard transfection protocol, the cells are
plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse
transfection protocol, cells are added directly to a plate containing the
transfection reagent/DNA mix and assayed on day 2 or 3. Because the cells are
added directly to the DNA, this process reduces the experimental time by one day
and allows for high-throughput transfection of DNA in a plate- or
microarray-format. For more information on reverse transfection including a protocol, read the PubHub article on the subject.

Stable Transfection

The goal of stable, long-term transfection is to isolate and propagate
individual clones containing transfected DNA that has integrated into the cellular
genome. Distinguishing nontransfected cells from those that have taken up
exogenous DNA involves selective screening. This screening can be accomplished by
drug selection when an appropriate drug-resistance marker is included in the
transfected DNA. Alternatively, morphological transformation can be used as a
selectable trait in certain cases. For example, bovine papilloma virus vectors
produce a morphological change in transfected mouse CI127 cells (Sarver
et al. 1981).

Before using a particular drug for selection purposes, you will need to
determine the amount of drug necessary to kill untransfected cells. This may vary
greatly among cell types. Consult Ausubel et al. 1995 for
additional information about designing experiments to test various drug
concentrations and determine the amount needed to select resistant clones (i.e.,
generate a kill curve).

When drug selection is used, cells are maintained in nonselective medium for
1–2 days post-transfection, then replated in selective medium containing the drug.
The use of selective medium is continued for 2–3 weeks, with frequent changes of
medium to eliminate dead cells and debris, until distinct colonies can be
visualized. Individual colonies can be isolated by cloning cylinders, selected and
transferred to multiwell plates for further propagation in the presence of
selective medium. Individual cells that survive the drug treatment expand into
clonal groups that can be individually propagated and characterized. For a
protocol to select transfected cells by antibiotics, see Stable Transfection (Section VII).

Several different drug-selection markers are commonly used for long-term
transfection studies. For example, cells transfected with recombinant vectors
containing the bacterial gene for neomycin phosphotransferase [e.g., pCI-neo
Mammalian Expression Vector (Cat.# E1841)] can be
selected for stable transformation in the presence of the neomycin analog G-418
(Cat.# V8091; Southern and Berg, 1982).
Similarly, expression of the hygromycin B phosphotransferase gene from the
transfected vector [e.g., pGL4.14 [luc2/Hygro] Vector
(Cat.# E6691)] will confer resistance to the
drug hygromycin B (Blochlinger and Diggelmann, 1984).

An alternative strategy is to use a vector carrying an essential gene that is
defective in a given cell line. For example, CHO cells deficient in dihydrofolate
reductase (DHFR) gene expression do not survive without added nucleosides.
However, these cells, when stably transfected with DNA expressing the DHFR gene,
will synthesize the required nucleosides and survive (Stark and Wahl, 1984). An
additional advantage of using DHFR as a marker is that gene amplification of DHFR
and associated transfected DNA occurs when cells are exposed to increasing doses
of methotrexate, resulting in multiple copies of the plasmid in the transfected
cell (Schimke, 1988).

Plasmid DNA is most commonly transfected into cells, but other macromolecules can
be transferred as well. For example, short interfering RNA (siRNA; Hong et
al. 2004; Snyder et al. 2004; Klampfer
et al. 2004), oligonucleotides (Labroille et
al. 1996; Berasain et al. 2003; Lin et
al. 2004), RNA (Shimoike et al. 1999; Ray and Das,
2004) and even proteins (Debs et al. 1990; Lin et
al. 1993) have been successfully introduced into cells via transfection
methods. However, conditions that work for plasmid DNA transfer will likely need to
be optimized when using other macromolecules. In all cases, the agent transfected
needs to be of high quality and relatively pure. Nucleic acids need to be free of
proteins, other contaminating nucleic acids and chemicals (e.g., salts from oligo
synthesis). Protein should be pure and in a solvent that is not detrimental to cell
health. For additional information on plasmid DNA quality, see DNA Quality and Quantity (Section III.D).

After cells are transfected, how will you determine success? Plasmids containing
reporter genes can be used to easily monitor transfection efficiencies and expression
levels in the cells. An ideal reporter gene product is one that is unique to the
cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally,
reporter gene assays are performed 1–3 days after transfection; the optimal time
should be determined empirically. For a discussion of luminescent reporter gene
options, see the Protocols and Applications Guide chapter on
bioluminescence reporters. A direct test for the
protein of interest, such as an enzymatic assay, may be another method to assess
transfection success.

In the case of siRNA, success may be measured using a reporter gene or assaying
mRNA (e.g., RT-PCR) or protein target levels (e.g., Western blotting). For additional
siRNA-specific reporter options, see the Protocols and Applications
Guide chapter on RNA interference.

If multiple assays will be performed, make sure the techniques you choose are
compatible with all assay chemistries. For example, if lysates are made from
transfected cells, the lysis buffer used ideally would be compatible with all
subsequent assays. In addition, if cells are needed for propagation after assessment,
make sure to retain some viable cells for passage after the assay.

With any transfection reagent or method, cell health, degree of confluency, number of
passages, contamination, and DNA quality and quantity are important parameters that can
greatly influence transfection efficiency. Note that with any transfection reagent or
method used, some cell death will occur.

Cells should be grown in medium appropriate for the cell line and supplemented
with serum or growth factors as needed for viability. Contaminated cells and media
(e.g., contaminated with yeast or mycoplasma) should never be used for transfection.
If cells have been compromised in any way, discard them and reseed from a frozen,
uncontaminated stock. Make sure the medium is fresh if any components are unstable.
Medium lacking necessary factors can harm cell growth. Be sure the 37°C incubator is
supplied with CO2 at the correct percentage (usually 5–10%)
and kept at 100% relative humidity.

As a general guideline, transfect cells at 40–80% confluency. Too few cells will
cause the culture to grow poorly without cell-to-cell contact. Too many cells results
in contact inhibition, making cells resistant to uptake of foreign DNA. Actively
dividing cells take up introduced DNA better than quiescent cells.

Keep the number of passages low (<50). In addition, the number of passages
for cells used in a variety of experiments should be consistent. Cell characteristics
can change over time with immortalized cell lines, and cells may not respond to the
same transfection conditions after repeated passages, resulting in poor
expression.

Plasmid DNA for transfections should be free of protein, RNA, chemical and
microbial contamination. Suspend ethanol-precipitated DNA in sterile water or TE
buffer to a final concentration of 0.2–1mg/ml. The optimal amount of DNA to use in
the transfection will vary widely, depending on the type of DNA, transfection
reagent, target cell line and number of cells.

You will need to optimize specific transfection conditions to achieve the desired
transfection efficiencies. Important parameters to consider are the charge ratio of
cationic lipid transfection reagent to DNA, amount of transfected nucleic acid, length
of time cells are exposed to the transfection reagent and presence or absence of serum.
Reporter genes are useful to determine optimal conditions. The transfection efficiency
achieved using any transfection reagents varies depending on the cell type being
transfected and transfection conditions used.

The amount of positive charge contributed by the cationic lipid component of the
transfection reagent should equal or exceed the amount of negative charge contributed
by the phosphates on the DNA backbone, resulting in a net neutral or positive charge
on the multilamellar vesicles associating with the DNA. Charge ratios of 1:1 to 2:1
TransFast™ Reagent:DNA have worked well with various cultured cells, but ratios
outside of this range may be optimal for other cell types or applications. See the
TransFast™ Transfection Reagent Technical Bulletin
#TB260 for more details.

The optimal amount of DNA or RNA will vary depending on the type of nucleic acid,
number of cells, culture dish size and target cell line used. For example, HEK-293
cells are optimally transfected with 0.25µg of pGL3-Control Vector
(Cat.# E1741) using TransFast™ Reagent at a 2:1
ratio in a 24-well plate. In contrast, the same cells are optimally transfected with
0.55µg of DNA using the FuGENE® HD Transfection Reagent at
a 3:1 ratio in the same well size. For other cell lines, we suggest testing the DNA
amounts given in Table 12.1.

Table 12.1. Suggested DNA Amounts to Use for Optimization.

Transfection Reagent

DNA Amount to Test

Reagent:DNA Ratios to Test

Culture Dish Size

FuGENE® 6
Transfection Reagent

0.04–0.2μg

4:1, 3.5:1, 3:1, 2.5:1, 2:1 and 1.5:1

96-well plate

FuGENE® HD
Transfection Reagent

0.04–0.2μg

4:1, 3.5:1, 3:1, 2.5:1, 2:1 and 1.5:1

96-well plate

TransFast™ Reagent

0.25, 0.50, 0.75, 1µg

2:1 and 1:1

24-well plate

Increasing the quantity of transfected DNA significantly may not yield better
results. In fact, if initial transfection results are satisfactory, a reduced DNA
quantity can be tested (while keeping the optimal reagent:DNA ratio constant). Often
a range of DNA concentration is suitable for transfection. However, if the DNA
concentration is below or above this range, transfection efficiencies will decrease.
If there is too little DNA, the experimental response may not be present. If there is
too much DNA, the excess can be toxic to cells. Calibrate the system using a test
plasmid with reporter gene function.

Traditionally, transfection reagents must be in contact with cells for some period
of time, then additional medium is added or the medium is replaced to help minimize
toxic effects of the reagent. The optimal transfection time depends on the cell line,
transfection reagent and nucleic acid used. For the
FuGENE® HD Transfection Reagent, which is one of the more
gentle methods of DNA transfection into cells, there is no need to add additional
medium or replace the medium after transfection.

For initial tests with liposomal reagents that require adding or replacing the
medium, use a one-hour transfection interval, and test transfection times of 30
minutes to 4 hours (Figure 12.4) or even overnight, depending on the reagent used.
Monitor cell morphology during the transfection interval, particularly when cells are
maintained in serum-free medium because some cell lines lose viability under these
conditions. The transfection time with the TransFast™ Reagent is usually
significantly shorter than that required with other cationic lipid compounds and can
be decreased to as little as 30 minutes with certain cell lines. In addition to
saving time, this shortened transfection time may significantly reduce the risk of
cell death.

Figure 12.4. Effect of transfection interval on transfection of CHO cells using
TransFast™ Reagent.CHO cells were transfected with 250ng of pGL3-Control DNA using
TransFast™ Reagent at a 2:1 reagent:DNA charge ratio for various times in
the absence of serum. All transfections were performed in 24-well plates,
and cell lysates were harvested 2 days post-transfection. The results
represent the mean of 6 replicates and are expressed as relative light units
(RLU) per well.

Transfection protocols often require serum-free conditions for optimal performance
because serum can interfere with many commercially available transfection reagents.
The TransFast™ and FuGENE® HD Transfection Reagents can be
used in transfection protocols in the presence of serum, allowing transfection of
cell types or applications that require continuous exposure to serum (e.g., primary
cells). Note that best results are obtained when variability is minimized among lots
of serum.

While many people use a single reporter gene for their experimental system, a
dual-reporter system has distinct advantages. A second reporter gene allows
expression to be normalized for transfection efficiency and cell number. Small
perturbations in growth conditions for transfected cells can dramatically affect gene
expression. A second reporter helps to determine if the effects are due to the
treatment of the cells or a response from the experimental reporter.

The Dual-Glo™ Luciferase Assay System (Cat.# E2920, E2940,
E2980) is an efficient means of quantitating luminescent signal from
two reporter genes in the same sample. In this system, firefly (Photinus
pyralis) and Renilla (Renilla
reniformis) luciferase activities are measured sequentially from a single
sample in a homogeneous format. In the Dual-Glo™ System, both reporters yield linear
assay responses (with respect to the amount of enzyme) and exhibit no endogenous
activity in experimental host cells. In addition, the extended half-life of the
reporter signals are ideal for use with multiwell assay formats.

The various Promega Renilla luciferase vectors can be used as
control vectors when
co-transfected with a firefly luciferase vector into which the promoter of
interest is cloned. Alternatively, the firefly vector may be used as the control
vector and the Renilla luciferase vector as the experimental
construct. In a co-transfection experiment, it is important to realize that
trans effects between promoters on co-transfected plasmids
can potentially affect reporter gene expression (Farr and Roman, 1992). This is
primarily of concern when either the control or experimental reporter vector or both
contain very strong promoter/enhancer elements. The occurrence and magnitude of such
effects will depend on several factors: 1) the combination and activities of genetic
regulatory elements present on the co-transfected vectors; 2) the amount and relative
ratio of experimental vector to control vector introduced into cells; and 3) the cell
type transfected.

To help ensure independent genetic expression of experimental and control reporter
genes, preliminary co-transfection experiments should be performed to optimize both
the vector DNA amount and ratio of co-reporter vectors. Because the Promega
Renilla luciferase vectors are designed for optimal
expression, it is possible to use very small quantities of these vectors to provide
low-level, constitutive co-expression of Renilla luciferase
activity. This means that the ratio of firefly and Renilla
luciferase vectors to test can range from 1:1 to 100:1 (or greater) to determine the
optimal expression. The key to a dual-reporter system is to maximize expression of
the experimental reporter while minimizing that of the control reporter. However, the
expression level of the control reporter should be three standard deviations above
background to be significant.

Additionally, experimental treatments may sometimes undesirably affect control
reporter expression. This compromises the accuracy of experimental data
interpretation; typically this occurs through sequences in the vector backbone,
promoter or reporter gene itself. For this reason, Promega offers different promoter
elements with either the same vector backbone, such as that of the pGL4.7 Vector
series, or a choice of vector backbones, available with the synthetic
Renilla luciferase vectors (phRL and phRG), to select the
most reliable co-reporter vector for your system. In fact, due to extremely
complicated cellular experimental conditions, testing several vectors is sometimes
required before finding the best internal control for a particular experimental
situation.

The strength of the promoter in your cell system is an important consideration. A
more moderately expressing promoter like thymidine kinase [TK; e.g.,
pGL4.74[hRluc/TK] Vector (Cat.#
E6921)] may be preferable to SV40 or CMV. Stronger promoters may
exhibit more trans effects, cross-talk or regulatory problems.
However, adjusting the ratio of experimental vector to control vector (e.g., using
100:1 or 200:1) may eliminate some of these issues.

The ProFection® Mammalian Transfection System—
Calcium Phosphate (Cat.# E1200) is a simple
system containing two buffers: CaCl2 and HEPES-buffered
saline. A precipitate containing calcium phosphate and DNA is formed by slowly mixing
a HEPES-buffered phosphate solution with a solution containing calcium chloride and
DNA. The DNA precipitate is distributed onto eukaryotic cells and enters cells
through an endocytic-type mechanism. Calcium phosphate transfection may be used to
produce long-term stable transfectants. It also works well for transient expression
of transfected genes and can be used with most adherent cell lines.

Special Usage Notes:

To increase transfection efficiency for some cell types, additional
treatments such as glycerol (Frost and Williams, 1978; Wilson and Smith, 1997),
dimethyl sulfoxide (DMSO; Lowy et al. 1978; Lewis
et al. 1980), chloroquine (Luthman and Magnusson, 1983)
and sodium butyrate (Gorman et al. 1983) may be added
during incubation with the calcium phosphate/DNA precipitate. These treatments
are thought to disrupt the phagocytic vacuole membrane, allowing the DNA to be
released to the cytoplasm (Felgner, 1990). Since each of these chemicals is
toxic to cells, transfection conditions for individual cell types, including
reagent concentration and exposure time, must be carefully optimized.

Citations

MC3T3-E1 (mouse preosteoblast) cells were plated at 5,000
cells/cm2 in 100mm dishes the day before
transfection. Cells were co-transfected with 15µg of plasmids expressing
either wildtype or dominant negative bone morphogenetic protein-2 (BMP-2)
and pSV-β-Galactosidase Control Vector at a 10:1 ratio in the presence of
serum for 16 hours using the ProFection®
Mammalian Transfection System—Calcium Phosphate. After the incubation,
the medium was changed and the expression assessed 72 hours
post-transfection by counting the number of β-galactosidase-positive
cells and assessing the expression of BMP-2 by Western blot
analysis.

Drosophila S2 cells were transfected with a U6
maxigene plasmid and a control firefly luciferase construct (derived from
the pGL2 Basic Vector) using the ProFection®
Mammalian Transfection System—Calcium Phosphate. Expression of the
maxigene was confirmed by isolating total RNA and performing primer
extension analysis with the aid of the Primer Extension System.
Luciferase assays were performed on an aliquot of cells after lysis with
Reporter Lysis Buffer.

The TransFast™ Reagent is supplied as a dried lipid film that forms multilamellar
vesicles upon hydration with water. The TransFast™ Transfection Reagent delivers
nucleic acid into eukaryotic cells in vitro and
in vivo (Bennett et al. 1997) and performs well with many
cell lines including NIH/3T3, CHO, HEK-293, K562, PC12, Jurkat and insect Sf9 cells.
The TransFast™ Reagent combines the advantages of cationic liposome-mediated
transfection with speed and ease-of-use to transfect cells for transient and stable
expression.

Special Usage Notes:

The TransFast™ Reagent can be used in the presence of serum, allowing
transfection of cell types that are serum-sensitive, such as primary cell
cultures.

Prepare the TransFast™ Reagent the day before transfection, because it needs
to be frozen before the initial use.

There are separate protocols to transfect adherent and suspension cells
using the TransFast™ Reagent.

Citations

To explore further the role of C/EBPb isoforms in regulating p53
expression during the cell cycle, the 1.7kb murine p53 promoter was
cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and
6629 (C/EBPb-null) cells were transfected using
0.1–0.75µg of pGL3-1.7-kb p53 promoter construct with or
without co-transfection of 0.25µg of C/EBPb-2 and 50ng of pRL-TK Vector
as an internal control. Twenty-four hours post-transfection, cells were
harvested and assayed for luciferase activity, normalizing reporter
activity to Renilla luciferase. Site-directed
mutagenesis was used to mutate or delete the –972/–953
cis-acting element carrying the C/EBPb-binding site
within the p53 promoter, and 0.1–0.75µg of each mutant construct was
transfected into Swiss3T3 cells with or without co-transfection of 0.25µg
of C/EBPb-2 and 50ng of pRL-TK Vector. The cells were harvested 24 hours
post-transfection and assayed for reporter activity, normalizing to
pRL-TK Vector activity.

The authors investigated the effect of various polymorphisms in the
dopamine transporter gene (SLC6A3) on susceptibility
to cocaine addiction. Genotyping of various polymorphisms in cocaine
abusers and control subjects revealed a potential association of the int8
VNTR with cocaine abuse. Seven alleles of the int8 VNTR were sequenced.
Various allelic sequences then were cloned into a modified phRL-SV40
Renilla luciferase reporter vector and
transfected into the mouse SN4741 cell line, which expresses the dopamine
transporter, and the effects on reporter activity were monitored.
Sequences of two alleles then were cloned into a pGL3 Promoter Vector
construct and transfected into JAP cells. The cells were challenged with
various amounts of cocaine or KCl and forskolin, and the effect on
reporter activity was monitored. The TransFast™ Reagent was used for
transfections at a 2:1 reagent:DNA ratio.

The FuGENE® HD Transfection Reagent
(Cat.# E2311) is a novel, nonliposomal formulation
to transfect DNA into a wide variety of cell lines with high efficiency and low
toxicity. The protocol does not require removal of serum (including up to 100% serum)
or culture medium and does not require washing or changing of medium after
introducing the reagent:DNA complex. Additionally, the
FuGENE® HD reagent supports transfection in chemically
defined media and does not contain any animal derived components. For more
information about transfection conditions using the
FuGENE® HD Transfection Reagent, visit the FuGENE® HD Protocol Database.

FuGENE® HD Transfection Reagent was used to
transiently transfect HeLa cells seeded on 12mm coverslips in 24-well
plates. Cells were seeded at a concentration of 5 ×
104 cells/well and transfected after 24 hours
using a 2:1 ratio of FuGENE® HD reagent to DNA
(4µl of reagent, 2µg of DNA). Transiently transfected cells were used for
confocal microscopy. For luciferase assays, HEK293 cells were seeded into
48-well plates at 40% confluency, and 24 hours later,
FuGENE® HD reagent was used to transfect
the cells with one of a variety of constructs.

The FuGENE® 6 Transfection Reagent
(Cat.# E2691) is a nonliposomal reagent that
transfects DNA into a wide variety of cell lines with high efficiency and low
toxicity. The protocol does not require removal of serum or culture medium and does
not require washing or changing of medium after introducing the reagent/DNA complex.
For more information about transfection conditions using the
FuGENE® 6 Transfection Reagent, visit the Transfection Assistant.

Citations

In this study, the FuGENE® 6 Transfection
Reagent was used to perform transient transfection of COS-7 cells.
Plasmids containing mutated or non-mutated copies of the estrogen
receptor gene ESR1 were transfected together with luciferase reporter
constructs containing estrogen response elements upstream of the
luciferase gene. Transactivation of the estrogen response element was
reduced in the mutated estrogen receptor compared with the nonmutated
receptor.

In this study, MCF7 human breast adenocarcinoma cells were transfected
with plasmid constructs using FuGENE® 6
Transfection Reagent. Cells were grown to 50% confluence and transfected
with 2µg of DNA at a 3:2 ratio of
FuGENE® 6 reagent:DNA.

Trypsinization Procedure to Remove Adherent Cells

Trypsinizing cells prior to subculturing or cell counting is an important
technique for successful cell culture. The following technique works consistently
well when passaging cells.

Materials Required:

1X trypsin-EDTA solution

1X PBS or 1X HBSS

adherent cells to be subcultured

appropriate growth medium (e.g., DMEM) with serum or growth factors or
both added

culture dishes, flasks or multiwell plates, as needed

hemocytometer

Prepare a sterile trypsin-EDTA solution in a calcium- and magnesium-free
salt solution such as 1X PBS or
1X HBSS. The 1X solution can be frozen and thawed for future use, but
trypsin activity will decline with each freeze-thaw cycle. The trypsin-EDTA
solution may be stored for up to 1 month at 4°C.

Remove medium from the tissue culture dish. Add enough PBS or HBSS to
cover the cell monolayer: 2ml for a 150mm flask, 1ml for a 100mm plate. Rock
the plates to distribute the solution evenly. Remove and repeat the wash.
Remove the final wash. Add enough trypsin solution to cover the cell
monolayer.

Place plates in a 37°C incubator until cells just begin to detach
(usually 1–2 minutes).

Remove the flask from the incubator. Strike the bottom and sides of the
culture vessel sharply with the palm of your hand to help dislodge the
remaining adherent cells. View the cells under a microscope to check whether
all cells have detached from the growth surface. If necessary, cells may be
returned to the incubator for an additional
1–2 minutes.

When all cells have detached, add medium containing serum to cells to
inactivate the trypsin. Gently pipet cells to break up cell clumps. Cells
may be counted using a hemocytometer and/or distributed to fresh plates for
subculturing.

Typically, cells are subcultured in preparation for transfection the next day.
The subculture should bring the cells of interest to the desired confluency for
transfection. As a general guideline, plate 5 × 104
cells per well in a 24-well plate or 5.5 × 105 cells
for a 60mm culture dish for ~80% confluency the day of transfection. Change cell
numbers proportionally for different size plates (see
Table 12.2).

Table 12.2. Area of Culture Plates for Cell Growth.

Size of Plate

Growth Areaa
(cm2)

Relative Areab

24-well

1.88

1X

96-well

0.32

0.2X

12-well

3.83

2X

6-well

9.4

5X

35mm

8.0

4.2X

60mm

21

11X

100mm

55

29X

aThis information was calculated for
Corning® culture dishes.

bRelative area is expressed as a factor of the total growth area of the
24-well plate recommended for optimization studies. To determine the
proper plating density, multiply 5 × 104 cells
by this factor.

High-quality DNA free of nucleases, RNA and chemicals is as important for
successful transfection as the reagent chosen. See the Protocols and
Applications Guide chapter on DNA
purification for information about purifying transfection-quality DNA.

In the case of a reporter gene carried on a plasmid, a promoter appropriate to the
cell line is needed for gene expression. For example, the CMV promoter works well in
many mammalian cell lines but has little functionality in plants. The best reporter
gene is one that is not endogenously expressed in the cells. Firefly luciferase,
Renilla luciferase, click beetle luciferase, chloramphenicol
aceyltransferase and β-galactosidase fall into this category. Vectors for all five
reporters are available from Promega. See the Reporter Vectors web page for
more information on our wide array of reporter plasmids.

In previous sections, we discussed factors that influence transfection success.
Here we present a method to optimize transfection of a particular cell line with a
single transfection reagent. For more modern lipid-based reagents such as the
FuGENE® HD Transfection Reagent, we recommend using
100ng of DNA per well of a 96-well plate at reagent:DNA ratios of 4:1, 3.5:1, 3:1,
2.5:1, 2:1 and 1.5:1. Figure 12.6 outlines a typical optimization matrix. When
preparing the FuGENE® HD Transfection Reagent:DNA complex,
the incubation time may require optimization; we recommend 0–15 minutes. Incubations
longer than 30 minutes may result in decreased transfection efficiency. See Technical
Manual #TM328.

Figure 12.6. Transfection optimization using the FuGENE®
HD Transfection Reagent.FuGENE® HD Transfection Reagent: DNA complexes
were formed by combining 2μg of pGL4.13[luc2/SV40]
Vector, enough FuGENE® Reagent to achieve the
indicated ratios and cell culture medium to a final volume of 100μl. To
HEK-293 cells in a 96-well plate (2 × 104
cells/100μl/well), 2μl, 5μl or 10μl of this complex was added per well.
Control wells contained untransfected cells, cells transfected with DNA only
(no FuGENE® HD Reagent) or cells transfected with
FuGENE® HD Transfection Reagent only (no DNA).
Firefly luciferase activity was measured using the ONE-Glo™ Luciferase Assay
System, and cell viability was determined using the CellTiter-Fluor™ Cell
Viability Assay. Luminescence and fluorescence were quantified using the
GloMax®-Multi+ Detection System, and results
are expressed as relative light units and relative fluorescence units (RFU),
respectively.

For traditional reagents, such as the TransFast™ Reagent, we recommend testing
various amounts of transfected DNA (0.25, 0.5, 0.75 and 1µg per well in a 24-well
plate) at two charge ratios of lipid reagent to DNA (2:1 and 4:1; see Figure 12.7 and
Technical Bulletin #TB260). This brief optimization can be performed using a
transfection interval of one hour under serum-free conditions. One 24-well culture
plate per reagent is required for the brief optimization with adherent cells (3
replicates per DNA amount).

A more thorough optimization can be performed to screen additional charge ratios,
time points and effects of serum-containing medium at the DNA amounts found to be
optimal during initial optimization studies. One hour or two hours for the
transfection interval is optimal for many cell lines. In some cases, however, it may
be necessary to test charge ratios and transfection intervals outside of these ranges
to achieve optimal gene transfer.

Both DEAE-dextran and calcium phosphate work well with larger cell cultures (e.g.,
100mm culture dish or T75 flask). General guidelines for DNA amount and time for
calcium phosphate-mediated transfection are given in Table 12.3.

Table 12.3. Guidelines for Calcium Phosphate Transfection.

Size of Culture Dish

Amount of DNA Transfected

Incubation Time with Transfection Complexa

60mm

6–12µg DNA

4–16 hours

100mm

10–20µg

4–16 hours

aIf the cells are sensitive to the reagent, incubate for no more than 4
hours. Incubation time can be longer but will need to be optimized for the
individual cell line.

Some transfection methods require removal of medium with reagent after incubation;
others do not. Read the technical literature accompanying the selected transfection
reagent to learn which method is appropriate for your system. However, if there is
excessive cell death during transfection, consider decreasing time of exposure to the
transfection reagent, decreasing the amounts of DNA and reagent added to cells,
plating additional cells and removing the reagent after the incubation period and
adding complete medium.

Many transient expression assays use lytic reporter assays like the
Dual-Luciferase® Assay System (Cat.#
E1910) or Bright-Glo™ Assay System (Cat.#
E2610) 24 hours post-transfection. However, the assay time frame can
vary (24–72 hours after transfection), depending on protein expression levels.
Reporter-protein assays use colorimetric, radioactive or luminescent methods to
measure enzyme activity present in a cell lysate. Some assays (e.g., Luciferase Assay
System) require that cells are lysed in a buffer after removing the medium, then
mixed with a separate assay reagent to determine luciferase activity. Others are
homogeneous assays (e.g., Bright-Glo™ Assay System) that include the lysis reagent
and assay reagent in the same solution and can be added directly to cells in medium.
Examine the reporter assay results and determine where the greatest expression
(highest reading) occurred. These are the conditions to use with your constructs of
interest.

Other assays include histochemical staining of cells (determining the percentage
of cells that are stained in the presence of the reporter gene substrate; Figure
12.8), fluorescence microscopy (Figure 12.9) or cell sorting if using a fluorescent
reporter like the Monster Green® Fluorescent Protein
phMGFP Vector (Cat.# E6421).

Figure 12.8. Histochemical staining of RAW 264.7 cells for β-galactosidase activity.RAW 264.7 cells were transfected using 0.1μg DNA per well and a 3:1 ratio
of FuGENE® HD to DNA. Complexes were formed for 5
minutes prior to applying 5μl of the complex mixture to 50,000 cells/well in
a 96-well plate. Twenty-four hours post-transfection, cells were stained for
β-galactosidase activity using X-gal. Data courtesy of Fugent, LLC.

Figure 12.9. Fluorescent microscopy of U-2 OS cells transfected with a
HaloTag®-NLS3 Vector
using the FuGENE® HD Transfection Reagent.U-2 OS cells were transiently transfected with 0.5µg of the
HaloTag®-NLS3 Vector,
which encodes the HaloTag® protein with three
copies of a nuclear localization signal (NLS), at a 3.5:1
FuGENE® HD Reagent:DNA ratio. Twenty-four
hours post-transfection, cells were labeled using the
HaloTag® TMR Ligand and the live-cell imaging
protocol described in the HaloTag
®
Technology: Focus on Imaging Technical Manual #TM260.
The resulting fluorescence was visualized by microscopy.

Assaying relative expression using the HaloTag®
technology provides new options for rapid, site-specific labeling of proteins in
living cells and in vitro. The ability to create labeled
HaloTag® fusion proteins with a wide range of optical
properties and functions allows researchers to image and localize labeled
HaloTag® protein fusions in live- or fixed-cell
populations and isolate and analyze HaloTag® protein
fusions and protein complexes. Several ligands are available for this system with new
options being added regularly. For more information on this labeling technology, see
the Protocols and Applications Guide chapter on cell labeling.

Optimization for stable transfection begins with successful transient
transfection. However, cells should be transfected with a plasmid containing a gene
for drug resistance, such as neomycin phosphotransferase (neo).
As a negative control, transfect cells using DNA that does not contain the
drug-resistance marker.

Forty-eight hours after transfection, trypsinize adherent cells and replate
at several different dilutions (e.g., 1:100, 1:500) in medium containing the
appropriate selection drug. For effective selection, cells should be
subconfluent since confluent, nongrowing cells are very resistant to the
effects of antibiotics like G-418.

For the next 14 days, replace the drug-containing medium every
3 to 4 days.

During the second week, monitor cells for distinct “islands” of surviving
cells. Drug-resistant clones can appear in 2–5 weeks, depending on the cell
type. Cell death should occur after 3–9 days in cultures transfected with the
negative control plasmid.

The following procedure may be used to determine the percentage of stable
transfectants obtained.

Note: The stained cells will not be viable after this procedure.

Materials Required:

methylene blue

methanol

cold deionized water

light microscope

After approximately 14 days of selection in the appropriate drug, monitor
the cultures microscopically for the presence of viable cell clones. When
distinct “islands” of surviving cells are visible and nontransfected cells have
died out, proceed with
Step 2.

Anson, D.S. (2004) The use of retroviral vectors for gene therapy-what are the risks? A review of
retroviral pathogenesis and its relevance to retroviral vector-mediated gene
delivery.
Genet. Vaccines Ther.2, 9.

Corning is a registered trademark of of Corning, Inc. FuGENE is a registered
trademark of Fugent, LLC. Geneticin is a registered trademark of Life Technologies,
Inc. Opti-MEM is a registered trademark of Invitrogen Corporation.

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